Acquired (somatic) mitochondrial DNA (mtDNA) mutations accumulate with age and reach high levels in dopaminergic neurons at early pathological stages in Parkinson's disease (PD). POLG ?mutator? mice have an accelerated accumulation of somatic mtDNA mutations and develop a premature aging phenotype at levels of mutations comparable to levels found in neurons in PD patients, demonstrating that somatic mtDNA mutations can be functionally significant. Furthermore, some patients with POLG mutations develop parkinsonism associated with ?-synuclein (?Syn) pathology and loss of dopaminergic neurons in the substantia nigra (SN). Based on these and other data, we hypothesize a ?two-hit? hypothesis whereby somatic mtDNA mutations contribute to mitochondrial dysfunction, and thereby exacerbate vulnerability to ?Syn. This may help to explain the dramatic rise with age in the incidence of PD. The overall goal of this proposal is to assess the relationship between somatic mtDNA mutations and vulnerability to ?Syn toxicity. We will do this using 2 strategies. First, we we will assess the impact of increased levels of somatic mtDNA mutations on ?Syn toxicity by assessing heterozygous and homozygous POLG mutator mice that overexpress double-mutant (A30P and A53T) ?Syn in dopaminergic neurons. Double-mutant ?Syn mice (dMut?Syn) develop a slowly progressive phenotype including striatal dopamine deficiency with behavioral deficits and loss of dopaminergic SN neurons. We predict earlier and more severe behavioral deficits, mitochondrial dysfunction, impaired mitophagy, more severe loss of striatal dopamine and dopaminergic terminals, and increased SN dopaminergic neuronal loss compared to ?Syn overexpression in the SN of WT mice. Second, we will perform stereotaxic SN injections of AAV-?Syn (wild-type or A53T) or a control vector (GFP-degron) in wild-type and heterozygous and homozygous POLG mutator mice. We again predict that somatic mtDNA mutations will exacerbate the phenotype associated with increased ?Syn expression, including the progressive death of neurons normally seen following AAV-?Syn injections, resulting in earlier and more severe deficits. These studies may yield the first experimental evidence that somatic mtDNA mutations, at levels relevant to early stages in PD, influence vulnerability to ?Syn toxicity, a result that may in part explain the dramatic rise in the incidence of PD with age. These studies also will have characterized a novel mouse model of dopaminergic neuronal degeneration that combines two mechanisms of pathophysiological relevance to PD.